The quantitative analysis of opioid ligand binding to receptor sites is important in the analysis of opiate drug or peptide action because it provides a measure of the critical primary interaction between the drug or endogenous opioid and the first in the series of tissue components which mediate the drug or ligand action. This interaction is the primary determinant of subsequent events initiated by receptor activation. In the current grant period, we have concentrated on analyzing the interactions of opioid agonists with specific types of opioid binding sites under conditions in which the consequences of receptor activation might be measured; that is, in the presence of physiological cations at normal body temperature. We now propose further studies which will extend our understanding of opioid receptor mechanisms. Under equilibrium binding conditions, we will look for interactions between different types of opioid binding sites by examining the changes in ligand interactions that occur when one type of binding site has been irreversibly inactivated. The nature of the regulation of agonist binding at each type of site by sodium and GTP will also be studied in more detail, and we will attempt to confirm that sodium regulation occurs at an intracellular site. Most evidence currently points to a critical role for both sodium and GTP in actions of opioids mediated through mu and delta type receptors. It is not yet clear if they plan an essential role in mediating kappa receptor directed events. We will also study ligand interactions with opioid binding sites at each type under nonequilibrium conditions, since true equilibria may seldom be achieved at the receptor under physiological conditions. In vitro studies of the consequences of opioid receptor activation are also proposed. Two actions of opioids will receive particular attention; inhibition of adenylate cyclase, and inhibition of neurotransmitter release, in rat or guinea pig cortex and corpus striatum. Here, a primary objective will be the determination of the receptor types mediating these effects.